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Cardiogenic and Myogenic Gene Expression in Mesenchymal Stem Cells After 5-Azacytidine Treatment

Objective: 5-Azacytidine is a hypomethylating agent that is used for the treatment of myelodysplastic syndrome. This histone modifier is widely employed and plays a nonspecific role in influencing the differentiation capability of stem cells. The ability of bone marrow mesenchymal stem cells to diff...

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Autores principales: Supokawej, Aungkura, Kheolamai, Pakpoom, Nartprayut, Kuneerat, U-pratya, Yaowalak, Manochantr, Sirikul, Chayosumrit, Methichit, Issaragrisil, Surapol
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Galenos Publishing 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3878477/
https://www.ncbi.nlm.nih.gov/pubmed/24385773
http://dx.doi.org/10.4274/Tjh.2012.0161
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author Supokawej, Aungkura
Kheolamai, Pakpoom
Nartprayut, Kuneerat
U-pratya, Yaowalak
Manochantr, Sirikul
Chayosumrit, Methichit
Issaragrisil, Surapol
author_facet Supokawej, Aungkura
Kheolamai, Pakpoom
Nartprayut, Kuneerat
U-pratya, Yaowalak
Manochantr, Sirikul
Chayosumrit, Methichit
Issaragrisil, Surapol
author_sort Supokawej, Aungkura
collection PubMed
description Objective: 5-Azacytidine is a hypomethylating agent that is used for the treatment of myelodysplastic syndrome. This histone modifier is widely employed and plays a nonspecific role in influencing the differentiation capability of stem cells. The ability of bone marrow mesenchymal stem cells to differentiate into cardiomyocyte- and myocyte-like cells after exposure to 3 different doses of 5-azacytidine has been evaluated and compared. The aim of the study was to optimize the effective dose of 5-azacytidine for promoting the cardiomyocyte and myocyte differentiation capabilities of human mesenchymal stem cells (MSCs). Materials and Methods: Human bone marrow aspirations were collected from healthy donors. MSCs were used for the study of mesodermal differentiation. MSCs were cultured to promote osteoblast differentiation and adipocyte differentiation. The evaluation of osteogenic or adipogenic properties was then performed through immunocytochemical staining. BMMSCs were trypsinized into single-cell suspensions and then prepared for flow cytometric analysis. The MSCs were treated with 5, 10, or 15 μM 5-azacytidine for 24 h and then cultured for 3 weeks. Total RNA was extracted from untreated and 5-azacytidine–treated cells. Troponin T and GATA4 antibodies were used as cardiogenic markers, whereas myogenin and MyoD antibodies were used as myocyte markers. Results: The morphology and growth rate of MSCs that were treated with any of the 3 doses of 5-azacytidine were similar to the morphology and growth rate of control MSCs. An immunofluorescence analysis examining the expression of the cardiac-specific markers GATA4 and troponin T and the skeletal muscle-specific markers MyoD and myogenin revealed that cells treated with 15 μM 5-azacytidine were strongly positive for these markers. Real-time RT-PCR results were examined; these amplifications indicated that there were higher expression levels of cardiac- and skeletal muscle-specific mRNAs in MSCs treated with 15 μm 5-azacytidine than in MSCs that had either been treated with lower doses of 5-azacytidine or left untreated. Conclusion: MSCs treated with 5-azacytidine demonstrated the capacity to differentiate into both cardiomyocytes and skeletal myocytes, and 15 μM 5-azacytidine could be the optimal dose of this drug. Other promoting factors should be examined to investigate the possibility of promoting the differentiation of MSCs into specific cell types. Conflict of interest:None declared.
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spelling pubmed-38784772014-01-02 Cardiogenic and Myogenic Gene Expression in Mesenchymal Stem Cells After 5-Azacytidine Treatment Supokawej, Aungkura Kheolamai, Pakpoom Nartprayut, Kuneerat U-pratya, Yaowalak Manochantr, Sirikul Chayosumrit, Methichit Issaragrisil, Surapol Turk J Haematol Research Article Objective: 5-Azacytidine is a hypomethylating agent that is used for the treatment of myelodysplastic syndrome. This histone modifier is widely employed and plays a nonspecific role in influencing the differentiation capability of stem cells. The ability of bone marrow mesenchymal stem cells to differentiate into cardiomyocyte- and myocyte-like cells after exposure to 3 different doses of 5-azacytidine has been evaluated and compared. The aim of the study was to optimize the effective dose of 5-azacytidine for promoting the cardiomyocyte and myocyte differentiation capabilities of human mesenchymal stem cells (MSCs). Materials and Methods: Human bone marrow aspirations were collected from healthy donors. MSCs were used for the study of mesodermal differentiation. MSCs were cultured to promote osteoblast differentiation and adipocyte differentiation. The evaluation of osteogenic or adipogenic properties was then performed through immunocytochemical staining. BMMSCs were trypsinized into single-cell suspensions and then prepared for flow cytometric analysis. The MSCs were treated with 5, 10, or 15 μM 5-azacytidine for 24 h and then cultured for 3 weeks. Total RNA was extracted from untreated and 5-azacytidine–treated cells. Troponin T and GATA4 antibodies were used as cardiogenic markers, whereas myogenin and MyoD antibodies were used as myocyte markers. Results: The morphology and growth rate of MSCs that were treated with any of the 3 doses of 5-azacytidine were similar to the morphology and growth rate of control MSCs. An immunofluorescence analysis examining the expression of the cardiac-specific markers GATA4 and troponin T and the skeletal muscle-specific markers MyoD and myogenin revealed that cells treated with 15 μM 5-azacytidine were strongly positive for these markers. Real-time RT-PCR results were examined; these amplifications indicated that there were higher expression levels of cardiac- and skeletal muscle-specific mRNAs in MSCs treated with 15 μm 5-azacytidine than in MSCs that had either been treated with lower doses of 5-azacytidine or left untreated. Conclusion: MSCs treated with 5-azacytidine demonstrated the capacity to differentiate into both cardiomyocytes and skeletal myocytes, and 15 μM 5-azacytidine could be the optimal dose of this drug. Other promoting factors should be examined to investigate the possibility of promoting the differentiation of MSCs into specific cell types. Conflict of interest:None declared. Galenos Publishing 2013-06 2013-06-05 /pmc/articles/PMC3878477/ /pubmed/24385773 http://dx.doi.org/10.4274/Tjh.2012.0161 Text en © Turkish Journal of Hematology, Published by Galenos Publishing. http://creativecommons.org/licenses/by/2.5/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Supokawej, Aungkura
Kheolamai, Pakpoom
Nartprayut, Kuneerat
U-pratya, Yaowalak
Manochantr, Sirikul
Chayosumrit, Methichit
Issaragrisil, Surapol
Cardiogenic and Myogenic Gene Expression in Mesenchymal Stem Cells After 5-Azacytidine Treatment
title Cardiogenic and Myogenic Gene Expression in Mesenchymal Stem Cells After 5-Azacytidine Treatment
title_full Cardiogenic and Myogenic Gene Expression in Mesenchymal Stem Cells After 5-Azacytidine Treatment
title_fullStr Cardiogenic and Myogenic Gene Expression in Mesenchymal Stem Cells After 5-Azacytidine Treatment
title_full_unstemmed Cardiogenic and Myogenic Gene Expression in Mesenchymal Stem Cells After 5-Azacytidine Treatment
title_short Cardiogenic and Myogenic Gene Expression in Mesenchymal Stem Cells After 5-Azacytidine Treatment
title_sort cardiogenic and myogenic gene expression in mesenchymal stem cells after 5-azacytidine treatment
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3878477/
https://www.ncbi.nlm.nih.gov/pubmed/24385773
http://dx.doi.org/10.4274/Tjh.2012.0161
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